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Isolation and Characterization of Bacteria in a Toluene-Producing Enrichment Culture Derived from Contaminated Groundwater at a Louisiana Superfund Site

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Isolation and Characterization of Bacteria in a Toluene-Producing Enrichment Culture Derived from Contaminated Groundwater at a Louisiana Superfund Site Louisiana State University LSU Digital Commons LSU Master's Theses Graduate School August 2020 Isolation and Characterization of Bacteria in a Toluene-Producing Enrichment Culture Derived from Contaminated Groundwater at a Louisiana Superfund Site Madison Mikes Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_theses Part of the Civil and Environmental Engineering Commons, and the Microbiology Commons Recommended Citation Mikes, Madison, "Isolation and Characterization of Bacteria in a Toluene-Producing Enrichment Culture Derived from Contaminated Groundwater at a Louisiana Superfund Site" (2020). LSU Master's Theses. 5206. https://digitalcommons.lsu.edu/gradschool_theses/5206 This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. ISOLATION AND CHARACTERIZATION OF BACTERIA IN A TOLUENE- PRODUCING ENRICHMENT CULTURE DERIVED FROM CONTAMINATED GROUNDWATER AT A LOUISIANA SUPERFUND SITE A Thesis Submitted to the Graduate Faculty of the Louisiana State University and Agriculture and Mechanical College in partial fulfillment of the requirements for the degree of Master of Science in The Department of Civil and Environmental Engineering by Madison Colleen Mikes B.S., Louisiana State University, 2018 December 2020 1 ACKNOWLEDGEMENTS I would like to take the time to thank all of those who have supported and assisted me during my graduate program. First and foremost, I would like to thank Dr. Bill Moe for all of the time he has spent teaching me and mentoring me through my thesis work. I have learned so much in the past two years, and I am so grateful that I had the opportunity to participate in this research. I would also like to thank the members of my thesis committee Dr. John Pardue and Dr. Gary King for their contributions to my thesis. I would like to acknowledge my laboratory peers that have supported me during the past two years. My thanks goes to Morgan, Alexis, Sam, Emily, Chi, Maura, and Shane for assisting me with my project as well as being there to discuss ideas and plans for new experiments. All of the continuous support kept me motivated and excited to go to the lab every day. Additionally, thank you to Sam Reynolds for developing the toluene-producing enrichment cultures that were used for my cultivation experiments. I am so grateful for the support of my family and friends. Thank you Mom, Dad, and Kel for motivating me to do my best work. Matt and Alison, thank you for being there for me and letting me vent to you when I was stressed and overwhelmed. I appreciate all of the advice and support. You made my experience in Baton Rouge so much more memorable. I also would like to thank Dr. Ying Xiao for assistance with scanning electron microscopy. Finally, I would like to thank the Louisiana Board of Regents and a consortium of petrochemical companies for financial support of my research. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS........................................................................................................ ii LIST OF TABLES ................................................................................................................................ v LIST OF FIGURES ............................................................................................................................ vii ABSTRACT ........................................................................................................................................viii CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW .................................................. 1 1.1. Introduction ......................................................................................................................1 1.2. Literature Review .............................................................................................................1 1.3. Research Objectives ..........................................................................................................8 1.4. Thesis Organization ..........................................................................................................9 CHAPTER 2. CULTIVATION AND ISOLATION OF BACTERIA DERIVED FROM A TOLUENE-PRODUCING ENRICHMENT CULTURE ON SOLID MEDIA ............................. 10 2.1. Introduction .................................................................................................................... 10 2.2. Toluene-Producing Enrichment Cultures ......................................................................... 10 2.2. Medium Preparation and Inoculation .............................................................................. 14 2.3. Stock Solution Preparation .............................................................................................. 18 2.4. Experimental Design for Isolation and Identification of Bacteria ..................................... 20 2.5. Results and Discussion.................................................................................................... 30 2.6. Conclusion ...................................................................................................................... 41 CHAPTER 3. CHARACTERIZATION OF NOVEL AZOSPIRA ISOLATES ............................. 42 3.1. Background of the genus Azospira .................................................................................. 42 3.2. Materials and Methods .................................................................................................... 44 3.3. Media Preparation ........................................................................................................... 45 3.4. Comparative Testing ....................................................................................................... 46 3.5. Results ............................................................................................................................ 56 3.6. Discussion ...................................................................................................................... 66 CHAPTER 4. CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE RESEARCH.. 70 4.1. Conclusions .................................................................................................................... 70 4.2. Recommendations for future research ............................................................................. 72 APPENDIX A. REPRESENTATIVE SEQUENCES FROM ISOLATES .................................... 78 iii APPENDIX B. STRAIN AZ-3 ASSEMBELED SEQUENCE ....................................................... 83 REFERENCES ................................................................................................................................... 84 VITA .................................................................................................................................................... 91 iv LIST OF TABLES Table 1.1. Phylogenetically related subdivision 1 Acidobacteria based on 16S rRNA gene sequences and maximum likelihood algorithm presented in Myers and King (2016)……………6 Table 2.1. Groundwater well location from which the SR enrichment culture originated [data from Moe et al. (2018)]……………………………………………...…………………….10 Table 2.2. Volatile organic compound (VOC) concentrations measured in groundwater collected for establishment of enrichment cultures from well SP022 on April 24, 2017 [222 days after the fourth molasses injection, data from Moe et al. (2018)] ……………………12 Table 2.3. Geochemical parameters measured in groundwater collected for establishment of enrichment cultures from well SP022 on April 24, 2017 [222 days after fourth molasses injection, data from Moe et al. (2018)]…………………………………………………………..12 Table 2.4. Growth Media used to Isolate Colonies from the Toluene-Producing Enrichment Cultures…………………………………………………………………………………………..17 Table 2.5. Universal bacterial Primers Used for Isolates…………………………….…………..23 Table 2.6. Universal and “Koribacter”-specific Primer Sets Used for Plate Wash PCR….….….25 Table 2.7. Expected Amplicon Size from Primer Sets…………………………………………..26 Table 2.8. Additional Primers used for Acidobacteria-targeted PCR…………………………...26 Table 2.9. Thermal Program for all Primer Sets………………………………………………....28 Table 2.10. Strains Inoculated into Liquid PA2D to Assess for Toluene Production…………...29 Table 2.11. Dilution Level of Colonies Isolated after Various Days of Incubation……………..31 Table 2.12. Closest Representatives to Grouped Isolates Based on the Blastn Algorithm Using the NCBI Database…………..……………………………………………………………34 Table 2.13. Closest Validly Published Bacterial Species to Grouped Isolates as Determined from EZ-Taxon……………………...…………………………………………………………...35 Table 2.14. Top Blastn Match for Positive Control DNA Amplified with Acidobacteria-specific Primer Sets…...………………………………………………………….39 Table 3.1. Pairwise similarity between strains Az-1, Az-2, and Az-3 and the most closely related type strains in the EZBioCloud database………………………………………………...56 Table 3.2. Substrate Utilization Profile for all Strains. (+) positive, (-) negative………..………61 v Table 3.3. Presence of Cellular Fatty Acids in all Strains (those that represent <0.5% in all strains were omitted). All data was reported in this study except those marked with an “*” which was reported by Bae et al. (2007)…………………………………………………….65 Table
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